GSM7383991: T cells (unstim), BATF-RFP knockin, donor 2; Homo sapiens; RNA-Seq
RNA was isolated from 2e5 sorted CAR T cells by standard, TRIzol-based RNA precipitation method as follows. Cells were resuspended in 1 ml TRIzol (Ambion). Chloroform was added (200 µL) to this lysate and extensively vortexed to achieve a homogenous mixture; then, it was incubated for 3 minutes at room temperature before centrifugation at 14,000g at 4C for 15 min. Aqueous layer was collected from the top and transferred into a new tube (~550 µL), 1 µL GylcoBlue (Ambion) was added, and the RNA was precipitated with equal volume of 2-propanol for 20 min at room temperature. RNA precipitates were centrifuged at 16,000g for 15 min at 4C and supernatant was carefully discarded without disturbing the GlycoBlue-stained blue RNA pellet. RNA pellet was washed with 1 ml 75% EtOH and after the wash, dissolved in 30 µL nuclease-free water. RNA concentration was determined by nanodrop, and RNA quality was determined by Agilent Bioanalyzer. Approximately 50 ng RNA was reverse-transcribed to complementary DNA (cDNA) and second strand was synthesized by the Ovation RNA-seq System V2 (Tecan) according to the manufacturer's recommendations. Double-stranded DNA was subjected to isothermal amplification and was purified with Ampure XP beads. DNA was quantified by Qubit and 80 ng DNA was used for sequencing library construction with the Ovation Ultralow Library System V2 (Tecan) using 8 PCR cycles according to the manufacturer's recommendations.